ABSTRACT: Radiotherapy is widely used to treat various types of cancer; however, its effectiveness is frequently limited by severe adverse effects resulting from radiation-induced damage to normal tissues in the tumor environment. Due to the scale of this problem and the current lack of safe and effective countermeasures against radiation toxicity, development of such agents is of broad significance for human health and presents an outstanding business opportunity. The new experimental drug CBLB502 is an agonist of mammalian Toll-like receptor 5 (TLR5) derived from its natural ligand, bacterial flagellin. CBLB502 is a powerful radioprotectant that was shown to selectively protect normal tissues, but not tumors, from radiation damage. In a recent study, CBLB502 effectively suppressed radiation-induced oral mucositis in a mouse model of head and neck cancer radiotherapy, while not reducing the antitumor efficacy of the radiation treatment. In fact, CBLB502 had a direct growth-suppressive effect on TLR5-expressing tumors, apparently by inducing an antitumor immune response. The therapeutic efficacy of CBLB502 is limited, however, by neutralizing antibodies detected in the course of preclinical animal studies and human safety trials performed by Cleveland BioLabs, Inc. Preliminary studies by the investigators of this proposal point to the existence of a limited number of epitopes recognized by anti-CBLB502 neutralizing antibodies. The central hypothesis of the proposed study is that these epitopes can be mapped and effectively eliminated by structure-guided protein engineering to generate a CBLB502 variant(s) with reduced immunogenicity and sensitivity to neutralizing antibodies, but no change in biopotency. Detailed structural knowledge of CBLB502-TLR5 binding and signaling, which was recently acquired by this research team, will provide critical guidance for this project, which includes the following specific aims: (i) To identify and eliminate CBLB502's main neutralizing epitopes; (ii) To characterize the activity, immunoresistance and immunogenicity of deimmunized CBLB502 variants in mice; and (iii) To test the efficacy of selected deimmunized CBLB502 variants against mucositis and other adverse effects of radiotherapy in the mouse model of head and neck cancer. Successful accomplishment of these tasks should yield a new TLR5 agonist with improved therapeutic potential for entry into Phase II studies focused on its development for anticancer applications.